Project Summary MRSA and VRE are pathogenic to Caenorhabditis elegans nematodes. Based on these observations, we developed C. elegans-MRSA and C. elegans-VRE screening assays performed in 384-well plates that can be used to screen compound libraries, allowing the identification of compounds that prevent host killing that would not be detected in traditional in vitro screens for antibiotics. The ability to eliminate highly toxic compounds is particularly important for the identification of compounds with activity against bacterial membranes, as these compounds can be broadly toxic against membranes of prokaryotic and eukaryotic and toxicity is a bottle-neck for the identification of antimicrobial agents that act on the membrane. In the previous funding period, we developed an automated, high throughput C. elegans ? MRSA screening platform and screened 88,489 small synthetic molecules and obtained 257 verified hits. Among these 257 hits, we prioritized FDA approved and other well-studied compounds that we found target the bacterial membrane. Additionally, we completed a screen of 39,931 synthetic compounds using a C. elegans ? VRE infection model assay and obtained 135 verified hits, including 4 VRE inhibitory compounds that block the ability of VRE to form a persistent intestinal infection. Our current overall objective is to carry out translational studies to determine whether the compounds we have prioritized can be developed as efficacious antimicrobial compounds. We propose to determine the modes of action of compounds with activity against the membrane of MRSA (Aim 1) and prioritize anti- infective compounds that block VRE colonization of the C. elegans intestine (Aim 2). In Aim 3, we propose to take the first steps to further develop these compounds by generating analogs, evaluating basic PK/PD characteristics, and performing efficacy studies in mouse models. In summary, we have used C. elegans-based assays to identify novel non-toxic antimicrobial compounds some of which can kill bacterial cells that are in a non-planktonic form of growth. We anticipate that the proposed studies will allow the discovery of potentially novel targets for antimicrobial drug discovery and identify potential lead compounds with activity against multi-drug resistant Gram-positive pathogens.